HDAC1 Promotes Liver Proliferation in Young Mice via Interactions with C/EBPβ

Abstract

HDAC1 (histone deacetylase 1) regulates a number of biological processes in cells. Our previous studies revealed that HDAC1 inhibits proliferation of the livers in old mice. We have surprisingly observed that HDAC1 is also increased in young livers proliferating after partial hepatectomy (PH) and in human liver tumors. Increased levels of HDAC1 after PH lead to its interaction with a member of the C/EBP family, C/EBPβ, which is also elevated after PH. At early time points after PH, the HDAC1-C/EBPβ complex binds to the C/EBPα promoter and represses expression of C/EBPα. A detailed analysis of the role of HDAC1 and C/EBPβ proteins in the regulation of C/EBPα promoter showed that, whereas C/EBPβ alone activates the promoter, HDAC1 represses the promoter in a C/EBPβ-dependent manner. The inhibition of HDAC1 in the livers of young mice inhibits liver proliferation after PH, which is associated with high levels of C/EBPα. Consistent with the positive role of HDAC1-C/EBPβ complexes in liver proliferation, we have found that the CUGBP1-HDAC1-C/EBPβ pathway is activated in human tumor liver samples, suggesting that HDAC1-C/EBPβ complexes are involved in the development of liver tumors. The causal role of the CUGBP1-HDAC1 pathway in liver proliferation was examined in CUGBP1 transgenic mice, which display high levels of the CUGBP1-eIF2 complex. We have demonstrated that elevation of the HDAC1-C/EBPβ complexes in CUGBP1 transgenic mice reduces expression of C/EBPα and increases the rate of liver proliferation. Thus, these studies have identified a new pathway that promotes liver proliferation in young mice and might contribute to the malignant transformations in the liver.

Liver is a quiescent tissue that is able to completely regenerate itself in response to partial hepatectomy and after surgical resections (1). Despite intensive investigations of liver regeneration, the mechanisms that control the transition of the liver from quiescence to proliferation are not well understood. Recent studies revealed that epigenetics play a critical role in the regulation of many biological processes, including cancer. Alterations of the chromatin structure are controlled by chromatin remodeling proteins, including histone acetylases and histone deacetylases. A member of the histone deacetylase family, HDAC1 (histone deacetylase 1), is expressed in the liver and might be involved in the regulation of liver growth and differentiation. A number of publications have revealed that that HDAC1 plays a role in development of the tumors (2). Although a general hypothesis for the role of histone deacetylases in tumors is that oncoproteins deliver histone deacetylases to inappropriate transcriptional repression of certain genes, such as p21, several publications show that expression and activity of HDAC1 are increased in cancer cells. For example, expression of HDAC1 is increased in invasive carcinoma of the breast and in prostate cancer (3-5). Despite the established role of HDAC1 in the promotion of tumor formation in prostate and breast cancer, very little is known about the role of HDAC1 in the regulation of liver proliferation and in development of tumors in the liver.

HDAC1 does not bind to DNA directly and displays its functions via interactions with transcriptional factors. It has been shown that HDAC1 associates with Rb and Brm or Brg1 complexes and regulates certain promoters during cellular senescence (6, 7). Recent observations have revealed that HDAC1 cooperates with C/EBP family proteins in the regulation of adipocyte differentiation (8). We have recently found that HDAC1 is also cooperating with C/EBPα in the livers of old mice and that this cooperation is involved in the inhibition of liver proliferation (9), which has been documented by many studies (10-12). In this work, we have found that HDAC1 displays its biological activities in livers of young mice through interactions with C/EBPβ. In young livers proliferating after PH and in human tumors, HDAC1 forms HDAC1-C/EBPβ complexes and releases negative control of proliferation by repression of the C/EBPα promoter. Examination of young CUGBP1 TR2 mice revealed that the CUGBP1-mediated induction of HDAC1 and C/EBPβ increases rate of liver proliferation, which correlates with the repression of C/EBPα.

EXPERIMENTAL PROCEDURES

Human Liver Tumor Samples and Liver Regeneration—Mouse liver regeneration in young animals was performed as described in our previous publications (13-15). Briefly, two-thirds of the liver was removed, and the remaining portion was allowed to proliferate for 4, 8, 24, and 48 h after the surgery. Mice were sacrificed, and the liver was kept in the freezer at-80 °C. Generation of CUGBP1 TR mice was described in our previous study (16). In these studies, we have used 4-6-month-old CUGBP1 TR mice. Animal experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine (protocol AN-1439). Human liver samples were obtained as a part of an institutional review board-approved protocol, where tumor and normal sections are collected from resected samples. These samples were used in the studies described in our previous work (14).

Identification of HDAC1 mRNA in CUGBP1-RNPs—To determine the association of HDAC1 mRNA with CUGBP1, CUGBP1 was immunoprecipitated from quiescent and regenerating mouse livers or from human control and tumor sections of the livers. RNA was isolated from these IPs and reverse transcribed using oligo(dT) primer. The presence of HDAC1 was examined by PCR with specific primers. The sequence of these primers is as follows: forward, 5′-ATTCCTGCGTTCTATTCGCCCAGA-3′; reverse, 5′-TTAGCAGTTCCAGGATGGCCAAGA-3′. As the control, PEPCK mRNA was determined in the CUGBP1 IPs. A summary of three or four independent experiments is shown.

Isolation of Protein Extracts—Cytoplasmic and nuclear extracts were isolated as previously described (14, 15). Briefly, livers were homogenized in buffer A, containing 20 mm Tris-HCl, pH 7.5, 30 mm KCl, 10 mm β-mercaptoethanol, and inhibitors of phosphatases. Nuclei were spun down at 12,000 rpm for 15 min, and supernatant (cytoplasm) was kept in the-80 °C freezer. The pellet (nuclei) was treated with buffer B containing 20 mm Tris-HCl, pH 7.5, 0.42 m NaCl, 10 mm β-mercaptoethanol, 25% sucrose, 5 mm MgCl₂, and inhibitors of phosphatases. After a 30-min incubation on ice, nuclei were spun down at 12,000 rpm for 10 min, and supernatant (nuclear extract) was frozen and kept in a-80 °C freezer.

Western Blotting and Co-immunoprecipitations—50 μg of proteins were loaded on gradient 8-16 or 4-20% SDS-polyacrylamide gel (Bio-Rad). Proteins were transferred on a nitrocellulose membrane, and the membrane was blocked with 10% dry milk on TTBS for 1 h. The membrane was incubated with primary antibodies for 2-4 h, washed, and incubated with secondary antibodies for 1 h. After wash, the signals were detected by detection reagents (Amersham Biosciences). Protein loading was verified by a reprobe of the membranes with β-actin and by Coomassie stain. Antibodies to C/EBPβ (C19), C/EBPα (A144), CUGBP1 (B1), HDAC1, cyclin A, cyclin E, eIF2α, Cdk2, and Cdk4 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to β-actin are from Sigma. TrueBlot secondary antibodies and IP beads from Ebioscience were used for Western blotting and co-immunoprecipitation studies.

Chromatin Immunoprecipitation (ChIP) Assay—The chromatin immunoprecipitation assay was performed using the ChIP-IT kit (Active Motif). The chromatin solutions were isolated from livers of young and old animals, and DNA was sheared by enzymatic digestion according to the instruction manual. Antibodies against C/EBPβ, C/EBPα, HDAC1, deacetylated histone H3, and trimethylated histone H3 were added to each aliquot of precleared chromatin and incubated overnight. Protein G beads were added and incubated for 1.5 h at 4 °C. DNA was isolated and used for PCRs with primers covering C/EBPα binding sites within the C/EBPα and PEPCK promoters. The sequences of the primers for these promoters are described in our previous study (17). PCR mixtures were amplified for one cycle of 95 °C for 5 min, annealing temperature for primers (62 °C) for 5 min, and 72 °C for 2 min. Then PCR mixtures were amplified for 34 cycles of 95 °C for 1 min, annealing temperature for 2 min, and 72 °C for 1.5 min. PCR products were separated by 1.5% agarose gel electrophoresis or by 4% PAGE.

Examination of Liver Proliferation in CUGBP1 Transgenic Mice—Three approaches have been used for the studies of liver proliferation in CUGBP1 TR mice. The first approach examined expression of cell cycle proteins by Western blotting as described above. The second approach included calculations of mitotic figures. The livers of WT and CUGBP1 TR mice were stained with hematoxylin and eosin, and the number of mitotic figures was calculated. The data presented were obtained with three animals of each genotype. The third approach examined E2F-Rb family complexes using EMSA with DNA probe covering the E2F site within the DHFR promoter. Nuclear extracts were incubated with the DHFR probe in buffer containing 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 5 mm dithiothreitol, 10% glycerol, and 0.5 μg of salmon DNA per 10 μl. Antibodies to E2F4, Rb, and p130 were added in the reactions before the probe addition. The binding reactions were loaded on native 5% polyacrylamide gel. The gel was dried and exposed with x-ray film.

RESULTS

Elevation of HDAC1 and C/EBPβ in Young Proliferating Livers Leads to Formation of HDAC1-C/EBPβ Complexes—We have recently found that HDAC1 is elevated in livers of old mice and is involved in the inhibition of liver proliferation (9). To determine the role of HDAC1 in the inhibition of liver proliferation in young animals, we examined if HDAC1 levels are reduced in livers proliferating after partial hepatectomy. Surprisingly, examination of HDAC1 in livers of young mice after PH showed that HDAC1 was increased at 4-24 h after PH (Fig. 1A). To ensure a proper proliferative response of the liver, expression of cyclin A was examined. We found that cyclin A was increased at 48 h after PH and showed a normal proliferative response of the animals (Fig. 1A). Calculations of HDAC1 levels as ratios to β-actin revealed a 2.5-3-fold increase of HDAC1 at early time points after PH (Fig. 1B). Examinations of HDAC1 mRNA by RT-PCR showed that mRNA is not changed in livers of young mice proliferating after PH (data not shown). Thus, these studies demonstrated that protein levels of HDAC1 are increased in young livers proliferating after PH. Since the elevation of HDAC1 takes place on the protein level, we suggested that partial hepatectomy stimulus activates translation of HDAC1. Our recent studies showed that RNA-binding protein CUGBP1 binds to the 5′ region of HDAC1 mRNA and increases translation of HDAC1 in livers of old mice and in young CUGBP1 TR mice (9). Therefore, we examined if proliferating livers utilize this pathway. Since translational activities of CUGBP1 are displayed through interactions of CUGBP1 with eIF2 (18), we initially examined the CUGBP1-eIF2 path-way in young livers after PH. Examination of CUGBP1 and eIF2 showed that, although protein levels of CUGBP1 and eIF2α were not significantly altered, the CUGBP1-eIF2α complexes were increased at 4-8 h and then reduced at 48 h after PH (Fig. 1C). We next examined if HDAC1 mRNA is associated with CUGBP1 in livers after PH. CUGBP1 was immunoprecipitated from cytoplasmic extracts isolated from quiescent livers (0 time point) and from livers at 8 h after PH, and RNA was isolated from these IPs and examined by RT-PCR with primers to HDAC1 mRNA. As can be seen in Fig. 1D, HDAC1 mRNA was observed in CUGBP1 IPs from liver proteins isolated at 8 h after PH but not in IPs from proteins isolated at the 0 time point. This association was specific, since RT-PCR with primers to PEPCK did not detect this mRNA in CUGBP1 IPs. Thus, these studies suggested that elevation of HDAC1 protein after PH is mediated by the CUGBP1-eIF2 complex.

FIGURE 1.

Elevation of HDAC1 in young livers proliferating after partial hepatectomy leads to the formation of a HDAC1-C/EBPβ complex. A, expression of HDAC1 in mouse liver after partial hepatectomy. Nuclear extracts from livers at different time after PH were examined by Western blotting with HDAC1 Abs. The membrane was reprobed with cyclin A and with β-actin. B, bar graphs show levels of HDAC1 as ratios to β-actin as a summary of two experiments. C, translational CUGBP1-eIF2 complex is increased after partial hepatectomy. Top (Western), Western blotting was performed with cytoplasmic extracts isolated at different time points after PH with antibodies to CUGBP1 and eIF2α. Bottom (CUGBP1-IP), CUGBP1 was immunoprecipitated from cytoplasmic extracts, and eIF2α was determined in these IPs. Positions of eIF2α and IgGs are shown. D, HDAC1 mRNA is associated with CUGBP1 in proliferating livers. CUGBP1 was immunoprecipitated from quiescent (0 h) and proliferating (8 h after PH) livers, RNA was isolated from these IPs, and HDAC1 mRNA was determined by RT-PCR. H20, control reaction without DNA. RNA, RT-PCR with total RNA isolated from mouse liver. E, the levels of HDAC1 and C/EBPβ are elevated in livers proliferating after PH. Western blotting was performed with antibodies to C/EBPβ (LAP and LIP isoforms), HDAC1, and cyclin E using protein extracts isolated at different time points after PH. Filters were reprobed with Abs to β-actin to verify protein loading. F, the formation of HDAC1-C/EBPβ complexes in proliferating livers correlates with the inhibition of C/EBPα. HDAC1 was immunoprecipitated from nuclear extracts, and IPs were probed with Abs to C/EBPα and C/EBPβ. IgG, heavy chains of immunoglobulins. Bottom, expression of C/EBPα after PH in examined animals.

The induction of HDAC1 in young livers was observed at the initial steps of liver proliferation (4-24 h), suggesting that this elevation might be involved in the promotion of proliferation. Since HDAC1 binds to promoters through the interactions with transcription factors, we examined if HDAC1 might cooperate with a member of the C/EBP family, C/EBPβ. C/EBPβ was selected for these studies, since the expression of C/EBPβ is increased in livers proliferating after PH (19, 20), and this increase correlates the with elevation of HDAC1 after PH (Fig. 1C). In addition, expression of C/EBPβ after PH is also mediated by the translational CUGBP1-eIF2 complex (18). We next examined levels of HDAC1 and C/EBPβ in protein lysates used for these studies and found that HDAC1 and C/EBPβ were increased after partial hepatectomy (Fig. 1E). Given this correlation, interactions of C/EBP proteins with HDAC1 were examined using a co-IP approach. In quiescent livers, both C/EBPα and C/EBPβ were associated with HDAC1. However, at 4 and 8 h after PH, the amounts of HDAC1-C/EBPβ complexes were dramatically increased, whereas C/EBPα-HDAC1 complexes were not detectable (Fig. 1F). The reduction of C/EBPα after PH correlates with the elevation of the C/EBPβ-HDAC1 complexes. Since the mouse C/EBPα promoter contains the C/EBP site, this correlation suggested that HDAC1-C/EBPβ might down-regulate the C/EBPα promoter. This suggestion was further examined in a set of experiments described below.

The HDAC1-C/EBPβ Complex Occupies and Represses the C/EBPα Promoter in Proliferating Livers—To examine if the HDAC1-C/EBPβ complexes occupy the C/EBPα promoter at 4-8 h after PH, a ChIP assay was performed with primers covering the C/EBP site within the mouse C/EBPα promoter. These studies showed that C/EBPα and C/EBPβ are bound to the C/EBPα promoter in quiescent livers. In proliferating livers, however, the C/EBPβ-HDAC1 complex was observed on the C/EBPα promoter, whereas C/EBPα was not detectable (Fig. 2A). To examine if the recruitment of HDAC1 to the C/EBPα promoter represses the promoter, we determined modifications of histone 3 on the C/EBPα promoter. A ChIP assay with antibodies to Ac-Lys-9-H3 and tri-Me-Lys-9-H3 showed that, in quiescent livers, acetylated histone H3 was abundant on the C/EBPα promoter, but it was not detectable at 4 h after PH. On the contrary, tri-Me-Lys-9 histone H3 was bound to the promoter at 4 h after PH. This pattern of histone H3 modifications shows that the C/EBPα promoter is repressed after PH. In addition to C/EBPα, expression of PEPCK is also reduced after PH and is regulated by C/EBP proteins (20). Therefore, we have examined if PEPCK might be an additional target of the HDAC1-C/EBPβ complex. A ChIP assay showed that the interactions of C/EBP and HDAC1 proteins with this promoter in quiescent and proliferating livers are similar to those observed on the C/EBPα promoter and that the HDAC1-C/EBPβ complex occupies the promoter in proliferating livers (Fig. 2A). Examination of histone H3 modifications on the PEPCK promoter showed that Lys-9 of histone H3 is deacetylated and trimethylated in regenerating livers. Thus, these data revealed that the HDAC1-C/EBPβ complexes are associated with C/EBPα and PEPCK promoters after PH and that this association leads to repression of the promoters and to down-regulation of C/EBPα and PEPCK (see below).

FIGURE 2.

C/EBPβ-HDAC1 complexes repress C/EBPα promoter. A, occupation of C/EBPα and PEPCK promoters by HDAC1-C/EBPβ complexes is increased in proliferating livers. A chromatin immunoprecipitation assay with C/EBPα and PEPCK promoters was performed using chromatin solutions from quiescent (0 h) and proliferating livers (4 h after PH). C/EBPα, C/EBPβ, HDAC1 (HD1), Ac-Lys-9-histone H3 (AcK9), and trimethyl-Lys-9-H3 (MeK9) were immunoprecipitated from the chromatin solutions and examined by PCR with primers covering the C/EBP site within the C/EBPα and PEPCK promoters. Con, PCR with chromosomal DNA; M, molecular weight markers; In, 1/100 of input; Ag, agarose beads. B, C/EBPβ-HDAC1 complex represses the C/EBPα promoter. The C/EBPα-luciferase reporter construct was co-transfected with C/EBPβ and HDAC1 individually and with C/EBPβ and increasing amounts of HDAC1 plasmid. Luciferase activity was calculated as a ratio to an internal control Renilla-luciferase. A summary of three independent experiments is shown. C, inhibition of the endogenous HDAC1 activates the C/EBPα promoter and increases the ability of transfected C/EBPβ to activate the promoter. HDAC1 was inhibited by siRNA, and C/EBPα promoter was co-transfected with C/EBPβ into the cells. The activity of the C/EBPα promoter was calculated as-fold induction relative to control (bar graph). Protein extracts from experimental cells were used for Western blotting to examine levels of HDAC1 and C/EBPβ (bottom image). D, siRNA to C/EBPβ inhibits C/EBPβ and reduces its transcriptional activity. Top, activity of the C/EBPα promoter in cells transfected with C/EBPβ and with C/EBPβ plus siRNA. Bottom, Western blotting with antibodies to C/EBPβ. The membrane was reprobed with β-actin. E, inhibition of endogenous C/EBPβ abolished the HDAC1-mediated repression of the C/EBPα promoter. The C/EBPα promoter was co-transfected with HDAC1 or with an empty vector in control cells and in cells with inhibited expression of C/EBPβ. The bottom image shows levels of C/EBPβ and HDAC1 in experimental cells determined by Western blotting. F, a hypothetical model for the repression of the C/EBPα promoter by HDAC1-C/EBPβ complexes.

The mouse C/EBPα promoter contains a C/EBP site, which is positively regulated by full-length C/EBP proteins (21). To determine if the HDAC1-C/EBPβ complexes repress the C/EBPα promoter, we cloned the mouse C/EBPα promoter into luciferase reporter vector and examined the effects of C/EBPβ and HDAC1 on the activity of the promoter. We first compared the effects of individual transfections of C/EBPβ and HDAC1 on the C/EBPα promoter and asked if simultaneous expression of these proteins might have a different effect. Fig. 2B shows that C/EBPβ alone activates the C/EBPα promoter; however, simultaneous transfections of C/EBPβ and HDAC1 led to inhibition of the C/EBPα promoter. HDAC1 alone also was able to inhibit the promoter, perhaps because of expression of endogenous C/EBPβ in Hep3B2 cells. Since transient transfection studies include overexpression of proteins, we asked if the HDAC1-C/EBPβ complexes inhibit the C/EBPα promoter at physiologically relevant concentrations. Therefore, we asked if the inhibition of endogenous HDAC1 or C/EBPβ would change the ability of the HDAC1-C/EBPβ complexes to repress the promoter. The expression of HDAC1 was inhibited in cells transfected with an empty vector and with vector expressing C/EBPβ. Interestingly, the inhibition of endogenous HDAC1 significantly increased activity of the C/EBPα promoter in control cells suggesting that the endogenous HDAC1-C/EBPβ complexes partially inhibited activity of the C/EBPα promoter (Fig. 2C). The inhibition of HDAC1 in cells transfected with C/EBPβ led to the higher activation of the promoter. We next asked if the inhibition of another component of the complex, C/EBPβ, would reduce the repression activity of HDAC1. The initial examinations of the siRNA to C/EBPβ showed that it efficiently inhibited expression of C/EBPβ and reduced its transcriptional activity toward the C/EBPα promoter (Fig. 2D). The inhibition of endogenous C/EBPβ in cells transfected with HDAC1 released the repression activity of HDAC1 (Fig. 2E). Taking together data of ChIP studies with the liver (Fig. 2A) and experiments in cultured cells, we conclude that C/EBPβ alone activates the C/EBPα promoter, whereas the C/EBPβ in the complexes with HDAC1 represses the C/EBPα promoter (Fig. 2F).

Inhibition of HDAC1 in the Liver Prevents a Reduction of C/EBPα after PH and Inhibits Liver Proliferation—To determine a possible role of C/EBPβ-HDAC1 complexes in the promotion of liver proliferation after PH, we inhibited expression of HDAC1 by siRNA (pKD-HDAC1-v4; Upstate Biotechnology, Inc.) and performed partial hepatectomy studies. To achieve high efficiency of delivery of siRNA to hepatocytes, we used the in vivo jetPEI-hepatocyte DNA transfection reagent kit from PolyPlus Transfection. With this approach, we obtained up to 60-70% efficiency of transfection into liver hepatocytes. Under these conditions, the expression of HDAC1 in the liver is inhibited to very low levels (less than 30%; see Fig. 3, A and B). A typical picture of immunostaining of HDAC1 in the liver is shown in Fig. 3A. As can be seen, partial hepatectomy caused a significant elevation of HDAC1 in control livers at 8 h after PH, but the majority of hepatocytes in the livers treated with siRNA to HDAC1 did not elevate HDAC1 after PH. We also found that the inhibition of HDAC1 in the liver for long time (3-4 days) led to a death of hepatocytes, perhaps due to necrosis. Therefore, the studies with siRNA-mediated inhibition of HDAC1 were performed within 48 h. As can be seen in Fig. 3, A and B, siRNA to HDAC1 reduced the levels of HDAC1, which remained low within 48 h after PH. We next examined HDAC1-C/EBPβ complexes in animals treated with control RNA and with siRNA to HDAC1 using a co-IP approach. These studies showed that HDAC1-C/EBPβ complexes were abundant in control mice at 8 and 36 h after PH, but these complexes were not detectable in livers of HDAC1 siRNA-treated mice (Fig. 3B). Western blotting studies revealed that the lack of HDAC1-C/EBPβ complexes led to a failure of the livers to reduce C/EBPα and PEPCK, whereas C/EBPβ was increased in both animal groups (Fig. 3, C and D). These data are consistent with modifications of histone H3, which are observed after partial hepatectomy on the C/EBPα and PEPCK promoters. Taken together, these studies showed that HDAC1 is a critical down-regulator of C/EBPα and PEPCK in proliferating livers and that the inhibition of HDAC1 leads to the failure to form HDAC1-C/EBPβ complexes. Since the HDAC1-C/EBPβ inhibited the C/EBPα promoter (Fig. 2), we suggest that high expression of C/EBPα and PEPCK after PH are due to the lack of the HDAC1-C/EBPβ complexes in siRNA-treated livers. Interestingly, protein levels of PEPCK and C/EBPα in quiescent livers with inhibited expression of HDAC1 are higher than those in quiescent control livers. We suggest that these high levels of PEPCK and C/EBPα might be supported by C/EBPβ, which activates the C/EBPα promoter in the absence of HDAC1 (see Fig. 2). To determine the consequences of the inhibition of HDAC1-mediated repression of C/EBPα, we have examined proliferative status of the liver by measuring levels of cell cycle proteins cyclin D1 and PCNA. Fig. 3E shows a Western blotting assay for cyclin D1 and PCNA, and Fig. 3, F and G, shows calculations of the levels of these proteins as ratios to β-actin. These studies showed that the expression of cyclin D1 and PCNA was increased in control animals at 48 h after PH; however, levels of cyclin D1 and PCNA were not increased in livers with inhibited HDAC1. Taken together, these studies showed that HDAC1 is involved in the inhibition of C/EBPα after PH and that a high level of C/EBPα in livers with inhibited HDAC1 correlates with reduced proliferation of the liver.

FIGURE 3.

Inhibition of HDAC1 reduces liver proliferation after PH. A, immunostaining of control and siRNA-treated livers with antibodies to HDAC1. Quiescent livers (0 time point) and livers at 8 h after PH were examined. B, inhibition of HDAC1 by siRNA blocks the formation of HDAC1-C/EBPβ complexes in the liver after PH. Partial hepatectomies were performed with control mice (injected with empty vector) and with mice injected with siRNA to HDAC1. Protein levels of HDAC1 and C/EBPβ were examined by Western blotting (Western). HDAC1-IP, HDAC1 was immunoprecipitated from nuclear extracts, and C/EBPβ was examined in these IPs. Positions of LAP and LIP isoforms of C/EBPβ are shown by arrows. IgG, heavy chains of immunoglobulins used for the IP. C, C/EBPα and PEPCK are not reduced after PH in livers with inhibited HDAC1. Expression of C/EBPα and PEPCK was examined by Western blotting. C/EBPα is expressed as two isoforms, 42 and 30 kDa. CRM, cross-reactive molecule on the C/EBPα filter serves as an additional loading controls. D, protein levels were calculated as ratios to β-actin and shown as percentage of 0 time point. E-G, high levels of C/EBPα in siRNA HDAC1-injected mice lead to a lack of activation of cell cycle proteins. E, expression of cyclin D1 and PCNA was examined by Western blotting. F and G, the levels of cyclin D1 and PCNA were calculated as ratios to β-actin.

The CUGBP1-HDAC1-C/EBPβ Pathway Is Activated in Human Liver Tumors—Given observations that the activation of the CUGBP1-HDAC1-C/EBPβ pathway is required for the liver proliferation after PH, we have asked if human liver tumors might activate this pathway to increase proliferation of hepatocytes. We obtained three samples from human liver tumor sections and from control healthy regions of the same livers in amounts sufficient for careful biochemical analysis. These samples were used for the studies described in this work. Cytoplasmic and nuclear extracts were isolated and examined for the expression of CUGBP1 (cytoplasmic proteins) and C/EBPβ and HDAC1 (nuclear extracts). We initially characterized samples regarding expression of proliferation markers and expression of CUGBP1. To confirm that the tumor sections have increased proliferation, we examined expression of cyclin D1 (a strong promoter of liver proliferation) and expression of p21 (an inhibitor of cell proliferation). A typical result of these experiments is shown in Fig. 4A. These experiments showed that cyclin D1 was dramatically increased in tumor sections, whereas p21 levels were reduced. Western blotting with antibodies to CUGBP1 showed that liver tumors increased protein levels of CUGBP1 up to 4-5-fold (Fig. 4, A and E) as well as amounts of hyperphosphorylated forms of CUGBP1, which migrate more slowly. To further examine the phosphorylation status of CUGBP1, the phosphorylated proteins were isolated using the PhosphoProtein purification kit (Qiagen) and examined by Western blotting with antibodies to CUGBP1. The result of these studies is shown in Fig. 4B. As one can see, liver tumors contain large amounts of hyperphosphorylated CUGBP1 compared with those observed in healthy sections of the liver. Coomassie stain of the membrane showed that total amounts of phosphorylated proteins do not differ significantly between tumor and control samples. Thus, these studies showed that liver tumors activate CUGBP1 by hyperphosphorylation. Since the hyperphosphorylation of CUGBP1 in mouse livers leads to the formation of the translational CUGBP1-eIF2 complex, we determined if human liver tumors contain this complex. Co-IP studies have shown that the CUGBP1-eIF2 complex is abundant in tumor liver sections, but it is not detectable in healthy sections of the same livers (Fig. 4C).

FIGURE 4.

CUGBP1-HDAC1-C/EBPβ pathway is activated in human liver tumors. A, expression of cell cycle proteins and CUGBP1 in liver tumors. Western blotting was performed with antibodies to cyclin D1, p21, and CUGBP1 using proteins isolated from control and tumor livers. The CUGBP1 filter was reprobed with Abs β-actin. B, isolation of phosphorylated proteins from control and liver tumor samples. Top, Western blotting of isolated proteins with antibodies to CUGBP1; bottom, Coomassie stain of the gel loaded with identical amounts of proteins that have been used for Western blotting. C, the CUGBP1-eIF2 complexes are abundant in tumor sections of the liver. CUGBP1 was immunoprecipitated from cytoplasmic extracts of control and tumor sections of the liver and examined by Western blotting with Abs to eIF2α. Top (input), levels of eIF2 determined by Western blotting. CRM, cross-reactive molecule that binds nonspecifically to the beads and serves as a loading control. D, expression of C/EBPβ and HDAC1 is increased in liver tumor samples. Western blotting was performed with protein extracts isolated from healthy sections of the livers (two controls) and from liver tumor sections (three samples) with antibodies shown on the right. Western blotting with cyclin E was performed to ensure that tumor sections proliferate, whereas control samples do not. β-Actin control is shown for HDAC1 and CUGBP1 membranes and for C/EBPβ (LAP and LIP isoforms) and cyclin E. Coomassie stain shows loading of the proteins performed with an independent gel. E, levels of CUGBP1, HDAC1, and C/EBPβ-LAP were calculated as ratios to β-actin. F, HDAC1 mRNA is associated with CUGBP1 in human liver tumors. CUGBP1 was immunoprecipitated from extracts isolated from quiescent control and tumor livers, RNA was isolated from these IPs, and HDAC1 mRNA was determined by RT-PCR. H20, control with H₂O. RNA, total RNA from the human liver. RT-PCR with primers to PEPCK mRNA was used as the control. G, tumor liver sections contain high levels of HDAC1-C/EBPβ complexes. C/EBPβ was immunoprecipitated from nuclear extracts, and IPs were probed with Abs to HDAC1. IgG, heavy chains of immunoglobulins.

We next examined expression of translational targets of CUGBP1: C/EBPβ and HDAC1 in these tumor samples. Fig. 4D shows that the elevation of CUGBP1-eIF complexes correlated with the increase of HDAC1 and C/EBPβ in tumor samples. Interestingly, expression of all isoforms of C/EBPβ, FL, LAP, and LIP were elevated in tumor sections. This elevation is consistent with our observations that the formation of the CUGBP1-eIF2 complex in the mouse livers increases translation of all isoforms and that the CUGBP1-eIF2 complex also increases translation of these isoforms in a cell-free translational system (22). The reprobe of the C/EBPβ membrane with antibodies to cyclin E detected this S-phase-specific protein in all three tumor samples but not in healthy sections of the livers. Densitometric calculations showed that levels of HDAC1 are 2.5-3-fold higher in tumor sections, whereas the levels of LAP are 4-5-fold higher (Fig. 4E). Given a correlation between elevation of CUGBP1 and HDAC1, we asked if CUGBP1 might activate translation of HDAC1. For this goal, we tested the interactions of CUGBP1 with HDAC1 mRNA. CUGBP1 was immunoprecipitated from cytoplasmic extracts isolated from control and tumor sections of the liver, and mRNA was isolated from these IPs and examined by RT-PCR with primers to HDAC1 mRNA. Fig. 4F shows that HDAC1 mRNA is associated with CUGBP1 in tumor sections but not in healthy sections of the liver.

Since HDAC1 and C/EBPβ form complexes in mouse livers after PH, we asked if human tumor livers contain these complexes. Co-IP studies were performed and showed that C/EBPβ-HDAC1 complexes are abundant in tumor sections but are undetectable in healthy sections of the liver (Fig. 4G). Taken together, investigations of three liver tumor samples revealed that the CUGBP1-HDAC1-C/EBPβ pathway is activated in tumors and is likely to be involved in the development of tumors. We tried to perform chromatin immunoprecipitation studies to determine association of the HDAC1-C/EBPβ with target promoters; however, the amounts of tumor samples were not sufficient to perform these studies.

Elevation of HDAC1-C/EBPβ Complexes in Livers of Young CUGBP1 Transgenic Mice Leads to the Inhibition of C/EBPα and to an Increased Rate of Liver Proliferation—Investigations of liver proliferation after PH and human tumor liver samples suggested that translational increase of HDAC1 by the CUGBP1-eIF2 complex is involved in promotion of liver proliferation. To directly test this overall hypothesis, we examined young CUGBP1 transgenic mice that express high levels of the CUGBP1-eIF2 complex (16, 22). To examine if the CUGBP1-eIF2 complex was formed in our experimental animals, CUGBP1 was immunoprecipitated from protein extracts of young WT and CUGBP1 TR liver as well as from protein extracts isolated from old livers. The proteins from livers of old mice were used as a positive control (9). As can be seen in Fig. 5A, the amounts of the CUGBP1-eIF2 complexes were dramatically increased in CUGBP1 TR mice. This elevation is similar to that observed in livers of old mice, which contain high levels of CUGBP1 (22). We next determined expression of CUGBP1, C/EBPα, C/EBPβ, and HDAC1 in livers of WT and in livers of CUGBP1 TR mice. These studies showed that the elevated expression of CUGBP1 and the CUGBP1-eIF2 complexes leads to the increase of C/EBPβ and HDAC1, whereas expression of C/EBPα is reduced in CUGBP1 TR mice (Fig. 5B). Co-IP studies revealed that amounts of C/EBPβ-HDAC1 complexes were increased in CUGBP1 TR mice (Fig. 5C). To determine if the HDAC1-C/EBPβ complex occupies the C/EBPα promoter, a ChIP assay was performed with chromatin solutions from WT and CUGBP1 TR livers. As can be seen in Fig. 5D, the C/EBPα promoter was occupied by C/EBPα and C/EBPβ in livers of WT mice, whereas only the HDAC1-C/EBPβ complex was bound to the promoter in livers of CUGBP1 TR mice. This pattern of binding correlates with the reduction of C/EBPα and is consistent with the hypothesis that the HDAC1-C/EBPβ complex represses the C/EBPα promoter.

FIGURE 5.

Activation of CUGBP1-HDAC1-C/EBPβ pathway in CUGBP1 TR mice leads to the increased rate of liver proliferation. A, CUGBP1 TR mice contain high levels of the CUGBP1-eIF2 complex. CUGBP1 was immunoprecipitated from cytoplasmic extracts of young (Y), old, and young CUGBP1 TR (TR) mice, and the IPs were probed with antibodies to eIF2α. Top (input), amounts of eIF2α in cytoplasmic extracts used for IP. IgG, heavy chains of immunoglobulins detected on the eIF2 filter. B, expression of CUGBP1, HDAC1, C/EBPα, and C/EBPβ in WT and in CUGBP1 TR mice. Liver cytoplasmic extracts (for CUGBP1) and nuclear extracts were examined by Western blotting with antibodies shown on the right. Each membrane was reprobed with Abs to β-actin. C, HDAC1-C/EBPβ complexes are increased in CUGBP1 TR mice. C/EBPβ and HDAC1 were examined in HDAC1 and C/EBPβ IPs correspondingly. IgG, heavy chains of immunoglobulins. D, HDAC1-C/EBPβ complexes occupy the C/EBPα promoter in livers of CUGBP1 TR mice. A ChIP assay was performed with chromatin solutions isolated from livers of WT and CUGBP1 TR mice. The experiment was performed as described in the legend to Fig. 2A. E, CUGBP1 TR livers contain an increased number of mitotic figures. Hematoxylin and eosin staining of WT and CUGBP1 TR livers was performed, and the number of mitotic figures found per 1000 hepatocytes was calculated and is shown in bar graphs. F, expression of cell cycle proteins in livers of CUGBP1 TR mice. Western blotting with antibodies (shown on the right) was performed with nuclear extracts isolated from WT and CUGBP1 TR livers. CRM, cross-reactive molecule observed on a Cdk2 filter. G, amounts of p130-E2F complexes are reduced in livers of CUGBP1 TR mice. A gel shift assay was performed with nuclear extracts isolated from WT and from CUGBP1 TR livers. Antibodies to E2F4, Rb, and p130 (shown on the top) were added to the binding reactions. Positions of E2F-Rb complexes and free probe are shown.

We next examined proliferative status of the CUGBP1 TR livers using three approaches: examination of mitotic figures, measurement of cell cycle proteins, and examination of Rb-E2F complexes. Calculations of the number of mitotic figures showed that the mitotic figures are very rare in WT livers; however, CUGBP1 TR livers contained a significant number of hepatocytes showing mitotic figures (Fig. 5E). We next examined expression of cell cycle proteins, levels of which are low or undetectable in quiescent livers but are increased during proliferation. Fig. 5F shows that protein levels of cyclin D1, PCNA, Cdc2, and cyclin A were increased in livers of CUGBP1 TR mice. Examination of E2F-Rb family complexes in WT and in CUGBP1 TR mice using a gel shift assay with the DHFR probe showed that the livers of WT mice contained abundant E2F4 and E2F-p300 complexes-repressors; however, the amounts of these complexes were dramatically reduced in CUGBP1 TR livers (Fig. 5G). Taken together, these studies revealed that livers of CUGBP1 TR mice have an increased rate of proliferation. These data support the hypothesis that translational induction of HDAC1-C/EBPβ complexes by CUGBP1 in livers of young mice and in human liver tumors promotes liver proliferation.

DISCUSSION

Proliferation of the liver is controlled by a complex network of several signal transduction pathways (1). Although the activation of CUGBP1 after PH has been previously described, information regarding downstream targets of CUGBP1 in proliferating livers is still limited. In this work, we have found that “active” CUGBP1 binds to the 5′ region of HDAC1 mRNA in livers and increases expression of HDAC1 protein at earlier stages of proliferation after PH. Since protein levels of HDAC1 are also increased in aged livers, which have reduced proliferative capacities, similar induction of HDAC1 in young proliferating livers suggests that HDAC1 might have different targets in these two opposite settings. Searching for the new partners of HDAC1 in livers of young mice, we obtained evidence that HDAC1 cooperates with C/EBPβ in the elimination of a negative control of liver proliferation. First, these two proteins are increased in livers proliferating after PH and in livers of CUGBP1 transgenic mice. Second, examination of the HDAC1-C/EBPβ complexes showed that they are abundant in young proliferating livers and that these complexes occupy promoters of genes whose expression is reduced in proliferating livers, such as C/EBPα and PEPCK. Since C/EBPα is required for the inhibition of proliferation, its down-regulation is likely to be involved in the promotion of proliferation. Third, C/EBPβ-HDAC1 complexes are increased in CUGBP1 TR livers that have an increased rate of proliferation. In agreement with data from CUGBP1 TR mice, our experiments with siRNA-mediated inhibition of HDAC1 in livers of young mice showed a critical role of this protein in the regulation of liver proliferation after PH. In quiescent livers of young mice, HDAC1 is associated with both C/EBPα and C/EBPβ. After partial hepatectomy, however, C/EBPβ is a major partner of HDAC1, targeting HDAC1 to C/EBPα and PEPCK promoters and inhibiting transcription of these genes. Consistent with these data, previous studies have shown that C/EBPβ KO livers have reduced proliferation after PH and express high levels of PEPCK and G6 pase mRNAs (20). Taken together, these data suggested that HDAC1 promotes liver proliferation after PH through the formation of HDAC1-C/EBPβ complexes.

Given the critical role of the HDAC1-C/EBPβ complexes in proliferation of mouse liver, we suggested that development of human liver tumors might be associated with activation of the CUGBP1 and accumulation of the C/EBPβ-HDAC1 complexes. Examination of this hypothesis in human liver tumors requires significant amounts of the tissues for detailed molecular studies. We have obtained three samples of liver tumors and control sections from normal specimens of the same livers in amounts sufficient for biochemical analysis. Investigations of these specimens revealed that CUGBP1 is activated in tumor sections and that translational targets of CUGBP1-eIF2, C/EBPβ and HDAC1, are also elevated and form the C/EBPβ-HDAC1 complex. Consistent with the data in mouse livers, CUGBP1 is associated with HDAC1 mRNA in tumor sections but not in healthy sections of human livers. Although data from liver tumors are correlative, they are consistent with data obtained in mouse models in which a causal role of elevation of CUGBP1 and HDAC1 has been established. To test the overall hypothesis, we have utilized CUGBP1 TR mice, which contain high levels of translational CUGBP1-eIF2 complex, a major regulator of the HDAC1-C/EBPβ complexes. These studies revealed that the CUGBP1 TR livers contain high amounts of the HDAC1-C/EBPβ complexes and have an increased rate of proliferation. Note that CUGBP1 has several targets in the liver and activates translation of a dominant negative isoform of C/EBPβ, LIP (19, 22). We suggest that LIP might also contribute to the liver proliferation in CUGBP1 TR mice, since its expression strongly correlates with the proliferative status of many tissues.

In summary, our data in three models of proliferating livers suggest that transition of the liver from quiescence to proliferation requires activation of the CUGBP1-eIF2 complex and that this activation leads to the increase in HDAC1 and C/EBPβ and in formation of the HDAC1-C/EBPβ complexes (Fig. 6). Experiments with a ChIP assay revealed that these complexes occupy promoters of C/EBPα and PEPCK and repress the activity of these promoters, which leads to the reduction of levels of corresponding proteins. Experiments with siRNA-mediated inhibition of HDAC1 in livers of young mice proliferating after PH have clearly shown that the elevation of C/EBPβ-HDAC1 complexes is required for liver proliferation.

FIGURE 6.

Translational elevation HDAC1 and C/EBPβ is involved in the promotion of liver proliferation in young mice. Shown is a hypothetical model for the role of CUGBP1-mediated elevation of HDAC1-C/EBPβ complexes in promotion of liver proliferation.